Plgs software

A total of 100 μL (1 × 10¹⁸ PFU/mL) purified phage was diluted with 50 mM NH₄HCO₃. It was then treated with 100 mM DTT at 95°C for 1 h followed by 250 mM iodoacetamide at room temperature in dark for 45 min. The sample was then digested with Trypsin and incubated overnight at 37°C. The peptides were extracted in 0.1% formic acid and incubated at 37°C for 45 min. The solution thus prepared was centrifuged at 10,000 × g and the supernatant was vacuum dried and dissolved in 20 μL of 0.1% formic acid in water. 10 μL of it was subjected to ACQUITY UPLC BEH C18 column (Waters, United Kingdom) for separation of peptides; the peptides separated on the column were directed to Waters Synapt G2 Q-TOF instrument (Waters, United Kingdom) for MS and MS/MS analysis. The raw data was processed using MassLynx 4.1 WATERS. The individual peptides MS/MS spectra were matched to the database sequence for protein identification on PLGS software, Waters. The obtained spectrometry information was analyzed with PLGS software 3.0.2 (Waters, United Kingdom) using the National Centre for Biotechnology Information (NCBI) non-redundant database and the specific database created in this study based on the predicted CDSs of phage Sfin-1. The important parameter settings for the PLGS analysis were as follows: peptide mass tolerance (ppm), 50; fragment mass tolerance (ppm), 100; maximal missed cleavages, 2.

Ten microliters of the cleaned sample was injected onto a BHE C18 UPLC column for separation of peptides (Supplementary Table), followed by analysis on a Waters Synapt G2 Q-TOF instrument for MS and MS/MS with an ESI source. The raw data were analyzed to obtain the complete integrated sequence of the sample by MassLynx 4.1 WATERS, peptide editor software. The individual peptide MSMS spectra were matched to the database sequence with the help of PLGS software, WATERS. The instrument used for acquiring mass spectrometry data was UPLC connected with Waters Synapt G2 (QTOF). The parameters used for identification are already mentioned, such as peptide mass tolerance at the MS1 level of 50 ppm and fragment mass tolerance at the MS2 level of 100 ppm. During the processing of the sample, cysteine sites were modified to carbamidomethylated cysteine, and the methionine sites were prone to oxidation, which was considered a variable modification to the mass (55 (link)–57 (link)). Quantitive details are given in Table S2.

Raw data were generated and processed by MassLynx 4.1 (Waters). The individual peptide MS/MS spectra were matched to the UniProt Bos taurus reference proteome database sequence for protein identification on the PLGS software (Waters). The parameters used for the identification are as follows: peptide mass tolerance at the MS1 level, 50 ppm; fragment mass tolerance at the MS2 level, 100 ppm; minimum number of fragment matches for peptides, 2; minimum number of fragment matches for proteins, 5; minimum number of peptide matches for proteins, 1; and missed cleavages, 1. Carbamidomethyl on cysteine as fixed modification and oxidation of methionine and N-terminal acetylation were considered as variable modifications for database search. The score is calculated by the expression analysis extension of the PLGS software based on the relevance of the protein present in both samples being compared. Differentially expressed proteins were identified by calculating the fold change of expression values (log base2) with respect to the control samples. Differentially expressed proteins include the proteins with higher abundance (< −1 fold) and lower abundance (< −1 fold) in the treated sample. The unique proteins mentioned for each control and treated are the proteins that did not have any matching peptides or m/z values between the groups.